Chimeric proteins, e.g., proteins comprising biologically active molecules and at least a portion of an immunoglobulin constant region, possess a number of desirable attributes. These include stability, which results in longer in vivo halflife, ease of purification and ease of administration to a subject. The expression of chimeric proteins comprised of immunoglobulin constant regions linked to a protein of interest, or fragment thereof, has been described (see e.g. U.S. Pat. Nos. 5,480,981 and 5,808,029; Gascoigne et al. 1987, Proc. Natl. Acad. Sci. USA 84:2936; Capon et al. 1989, Nature 337:525; Traunecker et al. 1989, Nature 339:68; Zettmeissl et al. 1990, DNA Cell Biol. USA 9:347; Byrn et al. 1990, Nature 344:667; Watson et al. 1990, J. Cell. Biol. 110:2221; Watson et al. 1991, Nature 349:164; Aruffo et al. 1990, Cell 61:1303; Linsley et al. 1991, J. Exp. Med. 173:721; Linsley et al. 1991, J. Exp. Med. 174:561; Stamenkovic et al., 1991, Cell 66:1133; Ashkenazi et al. 1991, Proc. Natl. Acad. Sci. USA 88:10535; Lesslauer et al. 1991, Eur. J. Immunol. 27:2883; Peppel et al. 1991, J. Exp. Med. 174:1483; Bennett et al. 1991, J. Biol hem. 266:23060; Kurschner et al. 1992, J. Biol. Chem. 267:9354; Chalupny et al. 1992, Proc. Natl. Acad. Sci. USA 89:10360; Ridgway and Gorman, 1991, J. Cell. Biol. 115, Abstract No. 1448; Zheng et al. 1995, J. Immun. 154:5590). These proteins were all produced using recombinant technology (see, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed., Cold Spring Harbor Laboratory Press (1989); Ausubel et al. 1989, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y.).
Recombinant technology provides a fast and relatively inexpensive way to produce large quantities of chimeric proteins, however the technology is not without its limitations. For example large multi-domain proteins can be difficult to express recombinantly. Recombinant expression of chimeric proteins often results in a heterogenous product requiring extensive purification. Some chimeric proteins may be toxic to cells making their expression, difficult, if not impossible. Moreover, recombinantly expressed proteins can only be comprised of the naturally occurring 20 amino acids. Thus, only L-configuration amino acids are possible using recombinant methods. Expressing chimeric proteins comprised of non-naturally occurring amino acids, provides a way to generate analogs useful in studying protein function and inhibiting undesirable metabolic pathways. Alternatively, analogs comprising non-naturally occurring amino acids may be used in some cases to enhance the activity of desirable metabolic pathways. Lastly, chimeric proteins comprising both amino acids and another biologically active molecules, e.g., nucleic acids, small molecules, are impossible to express using recombinant technology alone.
Many of the limitations described above may be overcome using chemical synthesis alone or a combination of recombinant techniques and chemical synthesis. A number of traditional techniques for chemically synthesizing proteins, such as solid phase synthesis are known in the art, see, e.g., Merrifield, 1973, Chemical Polypeptides, (Katsoyannis and Panayotis eds.) pp. 335-61; Merrifield 1963, J. Am. Chem. Soc. 85:2149; Davis et al. 1985, Biochem. Intl. 10:394; Finn et al. 1976, The Proteins (3d ed.) 2:105; Erikson et al. 1976, The Proteins (3d ed.) 2:257; U.S. Pat. No. 3,941,763.
Recent improvements in the chemical synthesis of proteins include the advent of native chemical ligation. As initially described, native ligation provides for the rapid synthesis of large polypeptides with a natural peptide backbone via the native chemical ligation of two or more unprotected peptide segments. In native ligation none of the reactive functionalities on the peptide segments need to be temporarily masked by a protecting group. Native ligation also allows for the solid phase sequential chemical ligation of peptide segments in an N-terminus to C-terminus direction, with the first solid phase-bound unprotected peptide segment bearing a C-terminal alpha-thioester that reacts with another unprotected peptide segment containing an N-terminal cysteine. Native chemical ligation also permits the solid-phase ligation in the C— to N-terminus direction, with temporary protection of N-terminal cysteine residues on an incoming (second) peptide segment (see, e.g., U.S. Pat. No. 6,326,468; WO 02/18417). Native ligation may also be combined with recombinant technology using intein linked to a chitin binding domain (Muir et al., 1998, Proc. Natl. Acad. Sci. USA, 95:6705).
Because chimeric proteins comprised of a biologically active molecule and at least a portion of an immunoglobulin constant region possess the desirable attributes described above, there remains a continual need for methods of synthesizing these chimeric proteins that is rapid and offers greater flexibility in the types of chimeric proteins produced. These needs, at least, are satisfied by certain embodiments of the invention.